In 2003, an initial draft of the first human genome sequence was completed at a cost of approximately $300 million, excluding the expenses for technological infrastructure. This large expenditure has stimulated competition for a more cost effective method of sequencing polynucleotides. Some of the proposed methods for achieving low cost polynucleotide sequencing utilize wet chemistry-based PCR, synthesis, or ligation methods. However, these methods may face challenges including short reads, PCR-related cost and an undesirable level of errors. Another proposed method uses nano-edge arrays to detect the vibration of individual nucleobases excited by tunneling electrons. However, this method may face challenges related to the uncertainty of electron tunneling in solutions.
A nanopore device provides a highly confined space through which single stranded polynucleotides can pass while individual bases are interrogated consecutively at high throughput without amplification or labeling. One compelling advantage of nanopore sequencing is the prospect of using unamplified genomic DNA, obviating the need for fluorescent reagents, as well as cloning and amplification steps, and eliminating the need for polymerases and ligases during readout. See, Branton et al., Nature Biotech 26, 1146 (2008). Thus, the costs of nanopore-based sequencing methods are projected to be far lower than the approaches used today. However, regardless of how promising the nanopore technology may be, several key technological challenges must be addressed before nanopore sequencing can be brought to the market place.
There are two general types of nanopores: natural biopores (e.g., α-hemolysin), and man-made solid-state pores, such those in metal-oxide-semiconductor (MOS) devices. To date, several different modes have been explored to use nanopores to sequence DNA. One technique involves measuring ionic current blockades as single stranded DNA is driven through a nanopore, either a biopore or a solid-state pore. Thus far, however, none of the nanopores studied appears to have the correct geometry to detect one nucleotide at a time while the polymer is translocating through the pore.
An alternative approach has been to measure transverse tunneling currents or capacitance as single stranded DNA is driven through a solid-state nanopore. It has been proposed that tunneling currents through nucleobases may be able to distinguish among the four nucleobases. Currently, two different approaches are typically used to measure such transverse tunneling currents. The first approach is to measure the tunneling current between two metal electrodes passing through a nucleobase of a translocating single stranded DNA. See Di Ventra et al., Biophys J, 93, 10, 2384 (2007). The advantage of this approach is that it aims to resolve information regarding a single nucleobase. However, this approach also has challenges. Chiefly, optimal voltage bias and solution conditions must be determined and maintained to provide unambiguous nucleobase identification in solution. Furthermore, the device must assure that each base will assume a reproducible orientation and position on the collector probe while it is being interrogated because tunneling currents are exponentially sensitive to atomic scale changes of orientations and distances.
The second approach has been to form base-specific hydrogen bonds between chemically-modified metal electrodes and the nucleobases. See Lindsay et al., Nano Lett, 7, 12, 3854 (2007). A nanopore device having a pair of electrodes functionalized with probes, with one probe able to couple to the nucleotide's phosphate moiety while another probe couples with the nucleobases, has been used in a ‘sequence by recognition’ scheme to identify the nucleobases. Major challenges of this approach include the need to fit a set of five probes (one for the backbone and four for the bases) at the tip of each nano electrode, and the synchronization of the formation and cleavage of the matching hydrogen bonds during DNA translocation.
Nanopore DNA sequencing based on an MOS capacitor has also been attempted. See Timp et al., Bell Labs Tech J, 10, 3, 5 (2005). One advantage of a capacitor-based nanopore device is that it does not have the problems associated with electron tunneling. As single stranded DNA translocates through a nanopore consisting of a parallel-plate MOS capacitor, variation of the electrostatic potential in the pore polarizes the capacitor, resulting in voltage fluctuations on the two silicon plates. In an early trial of this approach, a voltage signal associated with DNA translocation was detected, but it was not possible to distinguish between nucleotides. The pore channel of a length about 40 nm can accommodate a segment of single stranded DNA with about 100 nucleobases, suggesting that the measured results were due to multiple nucleobases. The relatively long span of the MOS capacitor in such a nanopore channel is inherent in the complex nature of an MOS device.
As such, there remains a need in the art for improved nanopore-based sequencing methods. There also remains a need in the art for improved devices and methods for the sequencing of other polymers. Additionally, there remains a need in the art for improved devices and methods for the detection of analytes, particularly biological analytes.